This invention relates generally to polymer nanoencapsulation and, more particularly, to nanocapsule carriers, and a method of using these carriers to enhance and stabilize enzyme reactivity or the reactivity of other encapsulated bioactive molecules.
This invention deals with nanoencapsulation and nanocapsules, and a method of using these encapsulation systems to enhance protein/enzyme reactivity. This invention also deals with using polymer nanocapsules to protect proteins and enzymes from inactivation in a variety of harsh environments such as extreme temperatures and pH. In addition, this invention also provides a method for using nanoencapsulation to enhance protein/enzyme reactivity in organic solvents. Finally, the present invention also provides a method of using nanocapsules as controlled-release agents or carriers for drug, protein, and vaccine delivery. Protein/enzyme stabilization is of great interest to a variety of applications including medical diagnostics, bioremediation, environmental clean-up, biocatalysis, and protein delivery. For example, protein or enzyme based medical diagnostic kits that exhibit prolonged shelf-life could improve performance and significantly reduce costs. For environmental decontamination or clean-up applications, the proteins/enzymes utilized need to be able to withstand a variety of harsh environmental conditions. These conditions could range from extreme temperatures (xe2x88x9230 to 60xc2x0 C.), extreme pHs (pH 1-12), and exposure to both polar and non-polar organic solvents such as methanol, toluene, hexane, and gasoline. For protein delivery applications, the protein carriers or stabilizers have to meet even more stringent requirements. The ideal protein carriers have to be not only non-toxic and non-immunogenic, but also must be able to protect labile proteins against natural deterioration. Moreover, in reality, the larger protein carriers (i.e. greater than 5-7 xcexcm) are often rapidly cleared from blood by capillary filtration primarily in the lungs. The smaller carriers (i.e. less than 200 nm), although free to circulate through capillaries, still face attacks from the immune system, thus being removed from blood rapidly by phagocytosis. Therefore, those carriers that are capable of generating long-term blood circulation of protein drugs can provide numerous advantages such as enhancing the efficiency of controlled-release drugs, providing site specific protein delivery, as well as reducing the need for repetitive injections.
Currently, protein stabilization has mainly been achieved by: 1) microencapsulation (i.e. with liposomes or water-soluble polymers); 2) bioconjugation (i.e. covalently linking proteins with water-soluble polymers or simply crosslinking proteins to form stable particles); or 3) genetic modification (i.e. genetically altering the protein sequence to make it more stable). However, microencapsulation that utilizes lipid-based micelles often suffers problems such as poor solution stability (especially under extreme temperatures and pH) and difficulty in being freeze-dried. In addition, the size distribution of these micelles is also very broad. The polymer-based microencapsulation, although significantly improving the freeze-drying capability, has very poor solution stability since only physical interactions are present between polymers and proteins. On the other hand, by using polyethylene glycol or oxide (PEG or PEO) modified liposomes (i.e. stealth liposomes) or biodegradable/non-degradable particles (stealth particles), the protein stability can be significantly enhanced. However, the sizes of these carriers are still too large (i.e. in microns) for more efficient and accurate delivery purposes. The bioconjugation of protein molecules with different water-soluble polymers such as PEGs and PEOs may also enhance the stability of proteins. However, this approach is very labor intensive, and, in some cases, the process can denature the proteins resulting in significant activity loss. Through proper genetic modification, the shelf-stability of proteins can be improved dramatically. Unfortunately, in most cases, the protein activity or specificity has also dropped very substantially.
U.S. Pat. No. 5,714,166, entitled xe2x80x9cBioactive and/or Targeted Dendrimer Conjugates,xe2x80x9d disclosed potential drug carrier applications using dense star polymers. However, this class of polymers is too small for encapsulating large molecules such as proteins, and therefore, does not meet the objective of the present invention. U.S. Pat. No. 5,919,442 entitled xe2x80x9cHyper Comb-Branched Polymer Conjugates,xe2x80x9d disclosed using larger Combburst polymers as drug carriers. However, no surface functionalization and size effects were described or disclosed as provided by the present invention. In addition, no enabling in vivo protein delivery examples were reported. The preparation of these prior art dendritic polymers also requires a core molecule and well-defined branches that are often more costly to produce through multi-step syntheses, whereas the polymers of the present invention can be obtained by a simple xe2x80x9cone-potxe2x80x9d synthesis strategy to generate randomly branched molecular structures without the need of a core molecule.
At present, there is a need for the development of nanoencapsulated enzymes and other nanoencapsulated bioactive molecules having improved stability, improved in vivo delivery characteristics, and the ability to withstand harsh environmental conditions.
Accordingly, a primary object of the present invention is to provide a nanoencapsulation method for enzymes and proteins.
It is another object of the present invention to provide a method of using this nanoencapsulation approach to formulate more stable protein carrying systems.
It is yet another object of the present invention to use this nanoencapsulation approach to produce protein-carrying formulations that are stable in both aqueous and organic media, as well as extreme pHs and temperatures.
It is still another object of the present invention to use this nanoencapsulation approach to generate nanocapsules that are stable in blood circulation, so that the encapsulated protein can be released in vivo in a controlled manner.
It is a further object of the present invention to provide a simple, inexpensive method of producing nanoencapsulated proteins, enzymes, or other bioactive molecules.
It is another object of the present invention to provide nanocapsules in the 10-500 nm range having an appropriate core size for encapsulation and protection of enzymes or other bioactive molecules.
It is still another object of the present invention to provide nanocapsules having surface functionalization such that solubility of the nanocapsule and reactivity of the encapsulated bioactive molecule is controlled and improved.
Finally, it is another object of the present invention to provide nanocapsules whose structure and characteristics are temperature sensitive so that delivery of encapsulated proteins or other molecules can be controlled by temperature changes.
The foregoing and other objects and advantages of the present invention will hereafter become more fully apparent from the following detailed description. In the description, reference will be made to examples and drawings which form a part hereof, and in which is shown by way of illustration, and not limitation, certain preferred embodiments. Such description does not represent the full extent of the invention, but rather, the invention may be employed according to the full scope and spirit of the invention as defined in the appended claims.